This invention relates to a non-animal based mixture of chemical and physical elements that serves as a control for a quantitative buffy coat analysis which is performed in accordance with the patented xe2x80x9cQBCxe2x80x9d paraphernalia. More particularly, this invention relates to a centrifugible non-animal based mixture which simulates blood, and which contains blood cell and other constituent-equivalent components in known amounts, which mixture can be gravimetrically separated into its blood constituent-equivalent components so as to be used as a control which simulates blood and which can be used for checking the accuracy of blood analysis instruments, which instruments are marketed by Becton Dickinson and Company under the trademark xe2x80x9cQBCxe2x80x9d.
Quantitative buffy coat (xe2x80x9cQBCxe2x80x9d) analysis is a technique utilized for measurement of hematologic parameters which include a plastic insert or float disposed in a capillary tube. Values for hematocrit, platelets, hemoglobin concentration, total white blood cell count, percent granulocytes, and percent lymphocytes and monocytes can be derived from measurements of layers that develop in the xe2x80x9cQBCxe2x80x9d tube during centrifugation of anticoagulated whole blood. U.S. Pat. Nos. 6,262,799; 5,889,584; 5,888,184; 5,830,639; 5,132,087; 4,940,668; 4,695,553; 4,594,165; 4,567,754; 4,137,755; 4,091,659; 4,082,085; 4,077,396; and 4,027,660 relate to the xe2x80x9cQBCxe2x80x9d technology in general and are incorporated herein in their entirety.
Quality control is a fundamental component of clinical laboratory practice. It is critical for ensuring the validity of analytical results and, as such, is an area of focus of regulatory agencies. In the xe2x80x9cQBCxe2x80x9d technology field, given the fact that the accuracy of the expansion of the buffy coat layer, and its constituents, is a function of the float diameter and the tube bore diameter, the control must be able to confirm that the platelet layer, the granulocyte and lymphocyte/monocyte layers, and the total white blood cell layer are being accurately measured.
The ideal xe2x80x9cQBCxe2x80x9d control must thus confirm the accuracy of the following parameters: 1) that the sample mixing and tube filling technique, i.e. the fill meniscus and hematocrit are correct; 2) that the height of the various cell layers referred to above are correct; 3) that the float density is correct; and 4) that the sensing mechanism and software used by the reader to detect and measure the height of the different colored and fluorescent bands affecting the buffy coat parameters are correct.
A xe2x80x9cQBCxe2x80x9d hematology control is described in U.S. Pat. No. 5,811,303 granted Sep. 22, 1998 to Wayne L. Ryan. The xe2x80x9cQBCxe2x80x9d hematology control is presently being marketed by Streck Laboratories of Minneapolis, Minn. The Streck control consists of: a red cell part having fixed mammalian red blood cells; a granulocyte component made from stained and fixed mammalian granulocytes; a lymphocyte/monocyte component also made from variably fixed mammalian granulocytes; a polystyrene latex bead component for the platelet component; and an isotonic saline suspension media with approximately twelve chemical additives. Several aspects of the Streck Laboratories composition make it adequate, but less than ideal. The separation, fixation, and staining processes are costly and time consuming, and chemical additives are necessary for ensuring the stability and proper sedimentation of the layers, thus adding to the cost and complexity. Furthermore, the animal basedal basis of the mixture is responsible for its relatively short stability and a significant variability of results. In particular, significant degradation has been observed about one week after shipment and unpredictability in results has been observed relating to the different mammalian sources of the animal based components of the control, and the varying efficiency of the processing steps, such as fixation.
It would be highly desirable to provide a xe2x80x9cQBCxe2x80x9d control substance having the following characteristics, i.e.,: it would remain a stable emulsion after mixing for at least two minutes prior to being placed in a xe2x80x9cQBCxe2x80x9d tube; it would be stable for shipping for at least sixty days after preparation, preferably at ambient temperatures; it would be non-hazardous to humans during preparation and use; it would be low cost and relatively simple to manufacture; it would be easy to use requiring nothing more than simple mixing; and it would be stable, after opening, using, and reclosing, for at least one week. The control mixture of the present invention provides all of the aforesaid desirable characteristics.
We have devised an anticoagulated whole blood-simulating control mixture that solves the aforementioned problems and that provides the aforementioned desirable characteristics. The control mixture of this invention includes a combination of fluorescent and/or otherwise colored beads having different densities and/or sizes and/or surface chemistries, since the density, size and the surface chemistry of the beads each or all in any combination can contribute to the degree of separation of the beads in the centrifuged control mixture. The beads are preferably combined in the mixture with a suitable number of generally immiscible liquid phases which have different densities and which phases are made into an essentially stable emulsion at 1 g.
The emulsion preferably includes one or more surfactants that will cause the liquid phases to separate into defined regions in the tube when the emulsion is centrifuged in the sample tube. The regions in the tube into which the liquid constituents gravitate will correspond to the plasma layer region and the red cell layer region in an anticoagulated whole blood sample which is centrifuged in the same type of tube. In addition to the liquid constituents in the control mixture, the mixture also includes three types of beads which are present in known amounts, and which are differentiated by color, size and/or density from each other. There will be an uppermost bead layer which simulates the platelet layer in a centrifuged sample of blood; a second medial bead layer which simulates the leukocyte/monocyte layer of the blood""s buffy coat; and a third lowermost bead layer which simulates the granulocyte layer in the blood""s buffy coat.
The granulocyte-simulating bead layer will preferably have a higher affinity for the liquid phase which simulates the red cell layer than for the other liquid phase which simulates the plasma layer in the centrifuged control. Thus, in descending order in the centrifuged control mixture, the less dense liquid forms the uppermost layer (plasma); the least dense and largest diameter bead group forms the uppermost formed component layer (platelets) in the simulated buffy coat; the medial density and medial diameter bead group forms the medial formed component layer (lymphocytes/monocytes) in the simulated buffy coat; the most dense and smallest diameter bead group forms the lowermost formed component layer (granulocytes) in the simulated buffy coat; and the most dense liquid constituent forms the red cell layer in the simulated blood sample.
It is therefore an object of this invention to provide a non-animal based emulsion of immiscible liquids and bead sets which will simulate the various components of centrifuged anticoagulated whole blood when the emulsion is centrifuged in a blood analysis tube.
It is a further object of this invention to provide an emulsion of the character described wherein the immiscible liquids simulate the plasma and red cell components of the whole blood.
It is another object of this invention to provide an emulsion of the character described wherein the bead sets simulate the components of a centrifuged white cell layer in whole blood.
It is an additional object of this invention to provide an emulsion of the character described wherein the bead sets and liquids are present in the emulsion in controlled amounts so as to enable the emulsion to be used as a control to verify the accuracy of an instrument which is used to determine complete blood counts in centrifuged blood samples.